Rose fermented product as well as preparation method and application thereof

Abstract

The invention discloses a rose fermented product as well as a preparation method and application thereof. According to the invention, the fermentation process is optimized, the combination of saccharomycetes, acetic acid bacteria and lactic acid bacteria is innovatively adopted for sequential fermentation, the first fermentation is saccharomycetes fermentation, the second fermentation is acetic acid bacteria fermentation, and the last fermentation is lactic acid bacteria fermentation. The fermented product effectively reduces cytotoxicity, effectively inhibits the generation of reactive oxygen species (ROS), and has a protective effect on oxidative stress injury of cells; compared with similar products, the content of effective components (flavone and total phenols) and the oxidation resistance (DPPH free radical scavenging rate, ABTS free radical scavenging rate and O2-free radical scavenging rate) are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant fermentation, and in particular relates to a rose fermentation product and a preparation method and application thereof. Background Art

[0002] Rose (Rosa rugosa Thunb.) is a perennial deciduous shrub belonging to the genus Rosa of the Rosaceae family. Rose flowers are warm in nature, sweet and slightly bitter in taste, and enter the kidney and spleen meridians, with a long history of medicinal use. Rose flowers are rich in various bioactive substances, including volatile oils, polysaccharides, polyphenols, flavonoids, etc., which have antioxidant, antibacterial, anti-tumor and lipid-lowering effects.

[0003] After fermentation, plant raw materials can greatly extend the shelf life of related products and improve their flavor quality, and can enhance biological activities such as anti-oxidation, anti-inflammatory, hypolipidemic, and immune regulation. Therefore, fermentation can make the active ingredients of rose and other medicinal and edible plants more fully released, thereby increasing the concentration of active ingredients, improving the efficacy and safety.

[0004] Existing rose fermented products have some defects in fermentation methods and functions, including the single fermentation strain used, resulting in single metabolites and insufficient retention of active ingredients, etc. These problems limit the application scope and market competitiveness of rose fermented products. Summary of the invention

[0005] The purpose of the present invention is to provide a rose fermented product and its preparation method and use. The present invention optimizes the fermentation process and innovatively adopts a combination of yeast, acetic acid bacteria and lactic acid bacteria for sequential fermentation (the first fermentation is yeast fermentation, the second is acetic acid bacteria fermentation, and the last is lactic acid bacteria fermentation). The fermented product effectively reduces cytotoxicity, effectively inhibits the generation of reactive oxygen species (ROS), and has a protective effect on cell oxidative stress damage; compared with similar products, the content of effective ingredients (flavonoids and total phenols) and antioxidant properties (DPPH free radical scavenging rate, ABTS free radical scavenging rate, O2 - free radical scavenging rate).

[0006] The purpose of the present invention is achieved through the following technical solutions: A method for preparing a rose fermented product comprises the following steps: (1) Preparation of culture medium The roses are beaten with water to obtain rose fluid, and then reducing sugar is added until the sugar content of the culture medium is 10-20 ° Brix, and a protein raw material is added to obtain a culture medium; The water-adding and slurrying is preferably followed by heating at 60-80° C., which is conducive to more dissolution of the active ingredient; The protein raw material is preferably one or more of soy protein isolate, whey protein or wheat hydrolyzed protein, and the added amount is 0.5-2.5% (based on the mass of the rose); (2) Fermentation The yeast is introduced into the culture medium for the first fermentation, and then pasteurized to obtain the first fermentation product; the pasteurized acetic acid bacteria is introduced into the first fermentation product, and then the second fermentation is carried out, and then pasteurized to obtain the second fermentation product; the lactic acid bacteria is introduced into the second fermentation product, and then the third fermentation is carried out, and then pasteurized to obtain the final fermentation product; The present invention selects yeast, acetic acid bacteria and lactic acid bacteria for sequential fermentation based on their metabolic characteristics and synergistic effects, which can significantly improve the antioxidant effect and functionality of the rose fermented product.

[0007] Yeast can convert sugars in roses into alcohol and produce a variety of secondary metabolites (such as esters and alcohols) to provide substrates for subsequent fermentation. Acetic acid bacteria then convert alcohol into acetic acid and produce a variety of organic acids and antioxidants. Lactic acid bacteria further utilize the remaining substrates to achieve efficient utilization of the substrates. The metabolites of the three strains (such as alcohol, acetic acid, lactic acid, polyphenols, peptides, etc.) can work synergistically to significantly enhance the antioxidant effect.

[0008] Furthermore, in the first fermentation, the amount of yeast added is 1.5-2 g / kg culture medium, the fermentation temperature is 25-30° C., and the fermentation days are 8-10 days.

[0009] Furthermore, in the second fermentation, the acetic acid bacteria inoculation amount is 2-10%, the initial alcohol content is 3-7% vol, the temperature is 26-34° C., and the fermentation days are 1-5 days.

[0010] Furthermore, in the third fermentation, the lactic acid bacteria inoculation amount is 2-10%, the temperature is 32-40° C., and the fermentation days are 1-5 days.

[0011] The lactic acid bacteria are one or more of Lactobacillus plantarum, Lactobacillus bulgaricus, Streptococcus thermophilus and Lactobacillus rhamnosus; Preferably, the lactic acid bacteria are used after being acclimated with a culture medium containing rose fluid; The culture medium containing rose fluid is prepared by adding rose fluid into a basic culture medium; The amount of the rose liquid used, based on the weight of the rose, accounts for 10-20% of the basic culture medium; The basic culture medium can be MRS culture medium, LBS culture medium, etc.; The domestication is to gradually increase the rose sap content in the basic culture medium so that the lactic acid bacteria gradually adapt to the growth conditions of the rose sap, and the lactic acid bacteria are domesticated for at least 5 generations; The final fermented product can be further centrifuged and the centrifuged supernatant can be homogenized to obtain a purified rose fermented product.

[0012] The rose fermented product prepared by the method can be used for preparing food, health products and medicines.

[0013] Compared with the prior art, the present invention has the following advantages and effects: (1) The fermented rose product of the present invention can effectively reduce the cytotoxicity of roses. Compared with untreated SH-SY5Y cells, there is no significant difference in the survival rate of cells treated with different concentrations of the fermented rose product, indicating that they have no cytotoxicity to SH-SY5Y cells.

[0014] (2) The rose fermentation supernatant of the present invention can effectively inhibit the generation of reactive oxygen species (ROS) in cells, reduce the level of ROS in cells, and play a protective role on cells.

[0015] (3) The rose fermentation supernatant of the present invention has a protective effect on oxidative stress damage of SH-SY5Y cells caused by H2O2.

[0016] (4) The rose fermented product of the present invention has increased flavonoid and total phenol content compared to that before fermentation. During the entire fermentation stage, the flavonoid and total phenol content in the fermentation broth showed a trend of first decreasing and then increasing.

[0017] (5) Compared with the fermented rose product before fermentation, the DPPH free radical scavenging rate, ABTS free radical scavenging rate, O2 - In vitro antioxidant activity indicators such as free radical scavenging rate were improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure shows the change of flavonoid content during fermentation process of the embodiment.

[0019] Figure 2 The figure shows the change of total phenol content during the fermentation process of the embodiment.

[0020] Figure 3 The free radical scavenging rate comparison chart of the fermented products of the embodiment and the comparative example and the vitamin C control group (A: DPPH free radical scavenging rate; B: ABTS free radical scavenging rate; C: O2 - free radical scavenging rate).

[0021] Figure 4 It is a comparison chart of the effects of the fermented products of the embodiment and the comparative example on the survival rate of SH-SY5Y cells.

[0022] Figure 5 It is a comparison chart of the effects of the fermented products of the embodiment and the comparative example on the active oxygen level of SH-SY5Y cells.

[0023] Figure 6 The effects of fermented products from Examples and Comparative Examples on MDA damage induced by H2O2 in SH-SY5Y cells.

[0024] Figure 7 The effects of fermented products from Examples and Comparative Examples on GSH damage induced by H2O2 in SH-SY5Y cells. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0026] The rose variety used in the specific implementation method is double-petal dark red rose; the yeast is Angel Saccharomyces cerevisiae; Acetobacter pasteurianus ( Acetobacter pasteurianus GDMCC1.67), Lactobacillus plantarum ( Lactobacillus plantarum GDMCC1.140), Lactobacillus bulgaricus ( Lactobacillus bulgaricus GDMCC1.155), Streptococcus thermophilus ( Streptococcus thermophiles GDMCC1.1808), Lactobacillus rhamnosus ( Lactobacillus rhamnosus GG GDMCC1.325) can be obtained from Guangdong Microbiological Culture Collection Center.

[0027] Example A method for preparing a fermented rose product comprises the following steps: (1) Preparation of rose juice: fresh roses are added with sterilized water to make juice, heated at 80°C, cooled and filtered; (2) Preparation of culture medium: adding 4% (based on the mass of the rose) sucrose to the rose fluid to adjust the sugar content to 20 ° Brix; and adding 1.5% (based on the mass of the rose) soy protein isolate to prepare the culture medium; (3) First fermentation: Inoculate the yeast into the above culture medium at an inoculum of 2 g / kg and a temperature of 25°C for 10 days. After fermentation, pasteurize the culture medium. (4) Second fermentation: Pasteurized acetic acid bacteria are inoculated into the first fermented product. The pasteurized acetic acid bacteria inoculation amount is 6%, the initial alcohol content is 5% vol, the temperature is 30°C, the fermentation days are 1-5 days, and pasteurization is performed after the fermentation is completed; (5) Domestication of lactic acid bacteria: By gradually increasing the content of rose sap in the MRS medium, Lactobacillus plantarum gradually adapted to the growth conditions of rose sap, and domesticated Lactobacillus plantarum for 5 generations. The proportion of rose sap in the medium increased from 20% in the first generation to 100% in the fifth generation, with an increase of 20% in each generation. The fifth generation of domesticated Lactobacillus plantarum was used for subsequent fermentation; (6) Third fermentation: the domesticated Lactobacillus plantarum is inoculated into the second fermentation product, the inoculation amount of Lactobacillus plantarum is 6%, the fermentation temperature is 36°C, the fermentation time is 5 days, and pasteurization is performed after the fermentation is completed; (7) Centrifugation and homogenization: Centrifuge the third fermentation product at 4000 r / min for 20 min, homogenize the supernatant, and obtain the desired rose fermentation product.

[0028] Comparative Example 1 The unfermented rose flower product is prepared by the following steps: (1) Preparation of rose juice: fresh rose flowers are added with sterilized water to make juice, heated at 80°C, cooled and filtered; (2) Centrifugation and homogenization: Centrifuge the rose fluid at 4000 r / min for 20 min, homogenize the supernatant, and obtain a rose product.

[0029] Comparative Example 2 The sample of this comparative example is the fermented product obtained by the first fermentation of the embodiment.

[0030] Comparative Example 3 The sample of this comparative example is the fermented product obtained by the second fermentation of the example.

[0031] Comparative Example 4 The preparation of the rose lactic acid bacteria fermented product comprises the following steps: (1) Preparation of rose water: same as step (1) in Example 1; (2) Preparation of culture medium: same as step (2) in Example 1; (3) Domestication of Lactobacillus plantarum: same as step (5) in Example 1; (4) Fermentation: The domesticated Lactobacillus plantarum is inoculated into the culture medium for fermentation at a temperature of 36°C and sterilized after fermentation is completed; (5) Centrifugation and homogenization: Centrifuge the lactic acid bacteria fermentation product at 4000 r / min for 20 min, homogenize the centrifugal supernatant, and obtain the desired rose lactic acid bacteria fermentation product.

[0032] Comparative Example 5 Preparation of products fermented by rose yeast, acetic acid bacteria and lactic acid bacteria: (1) Preparation of rose water: same as step (1) in Example 1; (2) Preparation of culture medium: same as step (2) in Example 1; (3) Domestication of Lactobacillus plantarum: same as step (5) in Example 1; (4) Yeast, acetic acid bacteria, and lactic acid bacteria co-fermentation: yeast, acetic acid bacteria, and domesticated Lactobacillus plantarum were inoculated into the above culture medium at the same time, with the yeast inoculation amount of 1 g / kg, the pasteurized acetic acid bacteria inoculation amount of 3%, and the Lactobacillus plantarum inoculation amount of 3%. The temperature was 30°C and the fermentation was carried out for 10 days. (5) Centrifugation and homogenization: Centrifuge the lactic acid bacteria fermentation product at 4000 r / min for 20 min, homogenize the centrifugal supernatant, and obtain the desired rose lactic acid bacteria fermentation product.

[0033] Test Example 1 Flavonoid content during fermentation (1) Test method Drawing of standard curve: Weigh 5.0 mg of rutin standard, dissolve it in 70% ethanol, transfer it to a 50 mL volumetric flask and dilute to the mark to prepare a 0.1 mg / mL rutin standard solution. Accurately measure 1, 2, 3, 4, and 5 mL of rutin standard solution in a 10 mL colorimetric tube, add 70% ethanol solution to 2 mL, then add 0.3 mL of 5% sodium nitrite solution to each tube, mix well and let stand for 6 min, continue to add 0.3 mL of 10% aluminum nitrate solution, shake well and let stand for 6 min, finally add 4 mL of 4% sodium hydroxide solution and fill to the mark with water, shake well and let stand for 15 min, measure the absorbance at 510 nm and draw a standard curve. All samples were measured in parallel 3 times.

[0034] Determination of flavonoid content in samples: The fermentation broth was centrifuged at 4000 r / min for 10 min, an appropriate amount of supernatant was taken and diluted 10 times, 2 mL of the solution was taken into a 10 mL colorimetric tube, and the subsequent steps were the same as above.

[0035] (2) Test results The changes in flavonoid content during the fermentation process are shown in Table 1 and Figure 1 As shown. During the entire fermentation stage of yeast, acetic acid bacteria, and lactic acid bacteria fermentation, the flavonoid content in the fermentation broth showed a trend of first decreasing and then increasing. In the yeast fermentation stage, that is, the first 10 days of fermentation, the flavonoid content gradually decreased from 125.73±1.70 mg / L to 88.15±1.85 mg / L, and then increased with the progress of acetic acid fermentation and lactic acid fermentation, and finally exceeded the content before fermentation. The flavonoid content increased to 130.71±2.14 mg / L after the end of lactic acid fermentation.

[0036] Table 1 Changes in flavonoid content during fermentation of Example

[0037] Test Example 2 Total phenolic content during fermentation (1) Test method Drawing of standard curve: Weigh 5.0 mg of gallic acid standard and dissolve it in a small amount of anhydrous ethanol, add distilled water to make it 10 mL, and make a 0.5 mg / mL standard solution. Measure 0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8 mL of standard solution in a 10 mL volumetric flask and dilute it with distilled water to make the final concentration 0, 20, 40, 60, 80, 100 mg / L, 120, 140 mg / L. Take 0.5 mL of standard solution of different concentrations in a 10 mL colorimetric tube, add 0.5 mL of Folin phenol, mix well and react in the dark for 5 minutes, then add 2 mL of 15% sodium carbonate solution, dilute to the scale with distilled water, mix well, and react at room temperature in the dark for 1 hour. Measure the absorbance of each solution at 765nm, with the horizontal axis as the concentration of the standard solution and the vertical axis as the absorbance, and draw a standard curve.

[0038] Determination of polyphenol content of samples: The fermentation broth was centrifuged at 4000 r / min for 10 min, and an appropriate amount of supernatant was diluted 20 times. 0.5 mL of the solution was taken into a 10 mL colorimetric tube, and the subsequent steps were the same as above.

[0039] (2) Test results The changes in total phenol content during the fermentation process are shown in Table 2 and Figure 2 As shown. During the entire fermentation stage of yeast, acetic acid bacteria, and lactic acid bacteria fermentation, the total phenol content in the fermentation broth also showed a trend of first decreasing and then increasing, which was the same as the trend of flavonoid content. In the yeast fermentation stage, that is, the first 10 days of fermentation, the total phenol content gradually decreased from 2.25±0.075 g / L to 1.32±0.091 g / L, and then increased again with the progress of acetic acid fermentation and lactic acid fermentation, and finally exceeded the content before fermentation. The total phenol content increased to 2.86±0.055 g / L after the end of lactic acid fermentation.

[0040] Table 2 Changes in total phenol content during fermentation of Example

[0041] Test Example 3 Evaluation of the antioxidant function of fermented products in vitro (1) Test method ① DPPH free radical scavenging rate Preparation of DPPH solution (0.2 mmol / L): Weigh 0.0197 g DPPH, dissolve it in anhydrous ethanol and dilute to 250 mL. Take 2 mL of sample solution in a centrifuge tube, add 2 mL of DPPH solution, shake well and react in the dark for 15 min, and measure the absorbance A1 at 515 nm; take 2 mL of sample and 2 mL of anhydrous ethanol to mix and react, and measure the absorbance A2 at 515 nm; take 2 mL of anhydrous ethanol and 2 mL of DPPH to mix and react, and measure the absorbance A0 at 515 nm. The DPPH free radical scavenging rate is calculated as follows: DPPH clearance rate % = [1-(A1-A2) / A0] × 100% ② ABTS free radical scavenging rate Preparation of ABTS working solution: Take 0.2 mL of ABTS solution (7.4 mmol / L) and K2S2O8 solution (2.6 mmol / L), mix them evenly, react at room temperature in the dark for 12 h, dilute 40-50 times with anhydrous ethanol to make the absorbance at 734 nm 0.7±0.2, and store the diluted solution in a refrigerator at 4°C in the dark for later use.

[0042] Take 0.8 mL of ABTS working solution in a centrifuge tube, add 100 μL of sample solution (diluted 100 times), mix well, shake for 20 seconds, let stand for 6 minutes, and measure the absorbance A at 734 nm. Use distilled water as a control and measure the absorbance A0 under compatible conditions. The ABTS free radical scavenging rate is calculated as follows: ABTS free radical scavenging rate % = (A0-A) / A × 100% ③ O2-free radical scavenging rate Take three 10 mL test tubes, add 2.5 mL Tris-HCl solution (50 mmol / L) to test tube 1, then add 1 mL sample solution and 0.6 mL pyrogallol solution, mix well, place the test tube in a 25°C water bath for 20 min, finally add 0.5 mL HCl to terminate the reaction, and measure the absorbance of the solution at 320 nm. j ; In test tube 2, water is used to replace the pyrogallol solution, and the subsequent treatment is the same as before. The absorbance value of the solution is measured as A i ; Replace the sample solution with water in the three test tubes, and the subsequent treatment is the same as before, and the absorbance value A0 of the solution is measured. The O2- free radical scavenging rate is calculated as follows: O2- clearance rate % = (A j -A i ) / A0×100% (2) Test results The results of the in vitro antioxidant test are shown in Table 3. The scavenging rates of rose flower before and after fermentation and Vc solution on DPPH free radicals varied between 34.01% and 85.40%. The antioxidant capacity of Example 1 was the strongest (85.40%), followed by Comparative Example 1 (78.40%), Comparative Example 3 (72.29%) (such as Figure 3 As shown in A in FIG. 1 ). For the DPPH free radical scavenging ability of the rose probiotic fermentation product, each milliliter of Example, Comparative Example 2, Comparative Example 3, and Comparative Example 4 is equivalent to containing about 2.51, 2.03, 2.12, and 1.92 mg / mL of Vc.

[0043] In the ABTS test, the ABTS free radical scavenging rate ranged from 45.43% to 91.75% (e.g. Figure 3 The antioxidant capacity of Example 1 is the strongest (91.75%), followed by rose yeast and acetic acid bacteria fermentation products (Comparative Example 3, 79.68%).

[0044] The O2-free radical scavenging rates of each fermented product and Vc varied between 63.57% and 94.54%. The O2-free radical scavenging rate of Vc was 94.54%. Except for Vc, the antioxidant capacity of the embodiment was the strongest (86.26%), followed by comparative example 1 (71.3±2.68%). The in vitro antioxidant test results showed that the in vitro antioxidant capacity of the embodiment was the best compared with other comparative examples.

[0045] Table 3 In vitro antioxidant levels of examples and comparative examples

[0046] Test Example 4 Evaluation of cellular antioxidant function in fermented products (1) Test method Cell culture Human neuroblastoma cells SH-SY5Y frozen in liquid nitrogen were taken out and thawed quickly in a 37°C water bath. The cells were then centrifuged at 1000 r / min for 3 min, the supernatant was discarded, and the cells at the bottom of the centrifuge tube were gently blown away with DMEM complete culture medium. The cell suspension was transferred to a 25 cm2 cell culture flask and cultured at 37°C, 5% CO2.

[0047] When the cell density reaches 80%-90%, remove the old culture medium, wash the cells 2-3 times with PBS solution, then add 1mL 0.25% trypsin-EDTA cell digestion solution, digest for about 1 min, add 1mL DMEM complete culture medium to terminate digestion, blow down the cells at the bottom of the bottle with culture medium, centrifuge the cell suspension, remove the supernatant, add complete culture medium to resuspend the cells, and then passage or plate.

[0048] Cytotoxicity assay SH-SY5Y cell suspension (100 μL) was inoculated in a 96-well cell culture plate, and the culture medium was removed after culturing for 24 h, and the plates were washed 1-2 times with PBS. DMEM culture medium containing 1000, 500, 250, 125, 125, and 62.5 μg / L of Example, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Comparative Example 5 was added to each well, respectively, and after culturing for 6 h, the liquid in the well was removed, and the plates were washed 1-2 times with PBS, and DMEM culture medium containing 10 μl CCK-8 was added to each well, and the plates were cultured at 37°C for 30 min, and the absorbance at 450 nm was measured using an enzyme marker.

[0049] Cellular reactive oxygen species detection The level of intracellular ROS was detected using a reactive oxygen species (ROS) detection kit and a flow cytometer. SH-SY5Y cells were inoculated into a 6-well cell culture plate, and the culture medium was removed after culturing for 24 h, and the cells were washed 1-2 times with PBS. The culture medium containing 250 μg / L of Example, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Comparative Example 5 was added respectively, and incubated in a 37°C cell culture incubator in the dark for 6 h. The liquid in the cell culture plate was removed, and a complete culture medium containing a positive control (Rosup, 50 μg / mL) was added, and the cells were incubated in the dark for 30 min. One group used an equal amount of PBS instead of Rosup as a blank control. The liquid was removed, washed once with PBS, and 1 mL of the diluted H2DCFDA working solution was added, and the cells were incubated in the dark for 30 min. The cells were washed 1-2 times with serum-free culture medium and observed under a fluorescence microscope. The above experiment was repeated, the cells were collected into flow tubes and resuspended with PBS, the DCF fluorescence signal intensity was detected by FACS-Aria III flow cytometer, and the data were analyzed using FlowJo software.

[0050] Establishment of cell oxidative damage model SH-SY5Y cell suspension (100 μL) was inoculated in a 96-well cell culture plate. After culturing for 24 h, the culture medium was removed and washed 1-2 times with PBS. DMEM containing different concentrations of H2O2 was added to each well, where the H2O2 concentrations were 0, 200, 400, 600, 800, 1200, and 1400 μmol / L, respectively. After culturing at 37°C for 2 h, the survival rate of SH-SY5Y cells was also determined by the CCK-8 method. Based on the experimental results, the most suitable H2O2 concentration for establishing a cell oxidative damage model was obtained.

[0051] Determination of cellular MDA and GSH SH-SY5Y cells were inoculated in a 6-well cell culture plate, and the culture medium was removed after culturing for 24 h, and the cells were washed with PBS 1-2 times. The culture medium containing 250 μg / L of Example, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Comparative Example 5 was added respectively. After the drug addition treatment was completed, the liquid in the well was removed, and the cells were washed twice with PBS precooled at 4°C, and the culture plate was placed on ice. After adding cell lysis solution to each well, the cells were shaken well, and after the cells were lysed, they were carefully collected into a centrifuge tube, and the supernatant was collected after centrifugation at 12000 r / min for 15 min, and stored at -80°C for use.

[0052] When determining the intracellular MDA content, it is necessary to determine the intracellular protein concentration. Take bovine serum albumin BSA as the standard, prepare a 0.5 mg / mL solution, add 0, 2, 4, 6, 8, 12, 16, 20 μL of BSA solution to a 96-well plate, add PBS to 20 μL, prepare BCA working solution with A solution and B solution in the BCA kit at a ratio of 50:1, add 200 μL of BCA working solution to each well, react at 37°C in the dark for 30 min, measure the absorbance at 562 nm with an enzyme reader, and draw a protein standard curve based on the protein concentration and the corresponding absorbance value.

[0053] MDA content determination The intracellular MDA level was detected by thiobarbituric acid (TBA) method. MDA standard solution (1 mmol / L) was prepared into 1.563, 3.125, 6.25, 12.5, 25, 50, and 100 μmmol / L for later use. The detection reaction system is shown in Table 4.

[0054] Table 4 Cell MDA detection reaction system

[0055] After adding the test solution according to the table, mix well, heat at 100℃ for 15 min, cool to room temperature, and centrifuge at 1000 r / min for 10 min. Take 200 μL of supernatant and add it to a 96-well plate. Use an ELISA reader to measure the absorbance at 532 nm, and draw the MDA standard curve based on the measured absorbance of the standard solution. MDA content calculation formula.

[0056]

[0057] Where: X-MDA content per unit weight of protein (unit: nmol / mg protein) Cpr-protein concentration (unit: mg / L) A determination - absorbance value of the sample to be tested at 532 nm A Blank-Blank control absorbance at 532 nm A standard - absorbance value of the standard solution at 532 nm GSH content determination The GSH kit was used to determine the intracellular GSH content. The GSH standard solution (1 mmol / L) was prepared into 0, 5, 10, 20, 50, and 100 μmmol / L for later use. The detection reaction system is shown in Table 5.

[0058] Table 5 Cellular GSH detection reaction system

[0059] After adding the test solution according to the table, gently shake the plate to mix, let it stand for 5 minutes, measure the absorbance at 405 nm with an enzyme marker, and draw a GSH standard curve based on the measured absorbance of the standard solution. GSH content is shown in the formula:

[0060] Where: X-GSH content per unit weight of protein (unit: μmol / g) A determination - absorbance of the sample at 405 nm A Blank-Blank control absorbance at 405 nm A standard - absorbance value of the standard solution at 405 nm (2) Test results Cytotoxicity test results Figure 4 As shown, the rose fermented product of the present invention can effectively reduce the cytotoxicity of roses. When the concentration of the unfermented rose liquid (Comparative Example 1) is 1000 μg / L, the cell survival rate is only 72.61%, indicating that it is toxic to SH-SY5Y cells. Compared with untreated SH-SY5Y cells, there is no significant difference in the survival rate of cells treated with different concentrations of Example, Comparative Example 2, and Comparative Example 3, indicating that they have no cytotoxicity to SH-SY5Y cells.

[0061] The intracellular ROS level was determined by flow cytometry quantitative analysis. The test results are shown in Table 6 and Figure 5 As shown. After SH-SY5Y cells were treated with Rosup, the proportion of ROS cells in the positive control group increased from 9.01% to 90.5%; the proportion of ROS cells in the embodiment was 49.95%, which was 44.8% lower than that in the positive control group. The proportions of ROS cells in Comparative Example 2 and Comparative Example 3 were 58.93% and 52.59%, respectively, which were 34.9% and 41.9% lower than those in the positive control group. As the sequential fermentation of yeast, acetic acid bacteria, and lactic acid bacteria proceeded, the ROS content gradually decreased. Compared with other comparative examples, the proportion of active oxygen reduction in the embodiment was the largest, proving that it has a stronger antioxidant effect.

[0062] Table 6 The proportion of cells with ROS

[0063] The content of MDA and GSH in cells Figure 6 , Figure 7 As shown in Table 7. Compared with the control group, after H2O2 induced oxidative damage in SH-SY5Y cells, the MDA content in the cells increased significantly from 5.61 nmol / mg to 22.34 nmol / mg, while compared with the H2O2 damage group, the MDA content in the cells treated with Examples and Comparative Examples 1-5 decreased. The MDA content in the cells of the Example was 11.27 nmol / mg, which was the lowest compared with other comparative examples, indicating that it had the lowest lipid oxidation level and had a protective effect on oxidative stress damage of SH-SY5Y cells caused by H2O2.

[0064] Compared with the control group, the GSH content of cells treated with H2O2 was significantly reduced to only 4.83 μmol / L. Compared with the H2O2 damaged group, the GSH content of cells treated with Examples and Comparative Examples 1-5 increased, with the largest increase in Examples, which was 16.21±0.35 μmol / L, proving that the antioxidant capacity of cells treated with Examples was stronger than that of the other Comparative Examples.

[0065] Table 7 Contents of MDA and GSH in cells

[0066] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a fermented rose product, characterized in that The following steps are involved: (1) Preparation of culture medium The roses are beaten with water to obtain rose fluid, and then reducing sugar is added until the sugar content of the culture medium is 10-20 ° Brix, and a protein raw material is added to obtain a culture medium; The added amount of the protein raw material is 0.5-2.5% based on the mass of the rose; (2) Fermentation The yeast is introduced into the culture medium for the first fermentation, and then pasteurized to obtain the first fermentation product; the pasteurized acetic acid bacteria is introduced into the first fermentation product, and then the second fermentation is carried out, and then pasteurized to obtain the second fermentation product; the lactic acid bacteria is introduced into the second fermentation product, and then the third fermentation is carried out, and then pasteurized to obtain the final fermentation product; The fermentation days for the first fermentation are 8 to 10 days, the fermentation days for the second fermentation are 1 to 5 days, and the fermentation days for the third fermentation are 1 to 5 days.

2. The preparation method according to claim 1, characterized in that: The protein raw material is one or more of soy protein isolate, whey protein or wheat protein hydrolyzate.

3. The preparation method according to claim 1, characterized in that: In the first fermentation, the amount of yeast added is 1.5-2 g / kg culture medium, and the fermentation temperature is 25-30°C.

4. The preparation method according to claim 1, characterized in that: In the second fermentation, the acetic acid bacteria inoculation amount is 2-10%, the initial alcohol content is 3-7%vol, and the temperature is 26-34°C.

5. The preparation method according to claim 1, characterized in that: In the third fermentation, the lactic acid bacteria inoculation amount is 2-10% and the temperature is 32-40°C.

6. The preparation method according to claim 1, characterized in that: The lactic acid bacteria are one or more of Lactobacillus plantarum, Lactobacillus bulgaricus, Streptococcus thermophilus and Lactobacillus rhamnosus.

7. The preparation method according to claim 1, characterized in that: The lactic acid bacteria are used after being tamed in a culture medium containing rose fluid.

8. The preparation method according to claim 7, characterized in that: The culture medium containing rose fluid is prepared by adding rose fluid into the basic culture medium.

9. The preparation method according to claim 7, characterized in that: The domestication is to gradually increase the rose fluid content in the basic culture medium so that the lactic acid bacteria gradually adapt to the growth conditions of the rose fluid, and the lactic acid bacteria are domesticated for at least 5 generations.

10. The preparation method according to claim 8, characterized in that: The amount of the rose liquid used, based on the weight of the roses, accounts for 10-20% of the basic culture medium.

11. The preparation method according to claim 8, characterized in that: The basic culture medium is MRS culture medium or LBS culture medium.

12. A fermented rose product, characterized in that: The method is prepared by the method described in any one of claims 1 to 11.

13. Use of the fermented rose product according to claim 12 in the preparation of food, health products and medicines.

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